Functionally differentiating two neuronal nitric oxide synthase isoforms through antisense mapping: Evidence for opposing NO actions on morphine analgesia and tolerance

Several isoforms of neuronal nitric oxide synthase (nNOS) have been identified. Antisense approaches have been developed which can selectively down-regulate nNOS-1, which corresponds to the full-length nNOS originally cloned from the brain, and nNOS-2, a truncated form lacking two exons which is generated bq alternative splicing, as demonstrated by decreases in mRNA levels. Antisense treatment also lowers nNOS enzymatic activity, Downregulation of nNOS-1 prevents the development of morphine tolerance, Whereas morphine analgesia is lost in control and mismatch-treated mice given daily morphine injections for 5 days, mice treated with antisense probes targeting nNOS-1 show no decrease in their morphine sensitivity over the same time period, Conversely, an antisense probe selectively targeting nNOS-2 blocks morphine analgesia, shifting the morphine dose-response curve over 2-fold to the right, Both systems are active at the spinal and the supraspinal levels, An antisense targeting inducible NOS is inactive, Studies with N-G-nitro-L-arginine, which does not distinguish among NOS isoforms, indicate that the facilitating nNOS-2 system predominates at the spinal level while the inhibitory nNOS-1 system is the major supraspinal nNOS system. Thus, antisense mapping distinguishes at the functional level two isoforms of nNOS with opposing actions on morphine actions, The ability to selectively down-regulate splice variants opens many areas in the study of nNOS and other proteins.